Plant-based formulations for improving liver health

ABSTRACT

The present invention is directed to plant based formulations for improving liver health by protecting the liver from alcohol and chemical insults and/or by inducing phase II enzymes. Formulations according to the present invention include wasabi root fiber powder, artichoke leaf extract, asparagus dehydrate, kudzu root extract, oregano extract, schisandra berry extract, notoginseng (ethanol extract of  Panax notoginseng  root), sanchi (water extracts from  Panax notoginseng  root), Gegen root extract ( Pueraria omeiensis ), spinach dehydrate, or combinations thereof.

BACKGROUND OF THE INVENTION

The present invention is directed to unique formulations of plant-basedextracts that work synergistically to aid in good liver health. Morespecifically, the present invention relates to a unique formulation ofplant-based extracts that assist in protecting the liver from alcoholand chemically induced damage.

The liver is one of the hardest working organs in the body. Good liverfunction is important for balanced hormone levels, weight control andmaintenance, cholesterol levels, skin health and general health. Theliver serves as the body's clearing house and is responsible for themetabolism of a number of substances, including alcohol, and plays animportant role in the detoxification of toxins in the body. Phase IIenzymes are part of this detoxification process because they aid in theremoval of potential carcinogens from the body. As a result of itsfunction in the body, the liver is under constant attack and prone todamage from environmental toxins, impurities, alcohol, prescription andover-the-counter drugs. Many hepatotoxicants such as carbontetrachloride, nitrosamines, and polycyclic aromatic hydrocarbons aremetabolically activated by liver enzymes to form reactive, toxicmetabolites that cause injury to the liver in humans.

Thus, a formulation of plant-based extracts that aids in protecting theliver against alcohol and carbon tetrachloride insults would be useful.Additionally, a plant-based formulation that works to induce phase IIenzymes responsible for detoxifying the liver would be useful.

SUMMARY OF THE INVENTION

The present invention is directed to unique formulations that improveliver health by working to protect the liver from carbon tetrachlorideand alcohol insults. Formulations of the present invention have shownstrong protective abilities on human liver cells as measured by indicessuch as 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide,which is a tetrazolium salt, (“MTT”) and lactase dehydrogenase (“LDH”).In addition, these extracts and their combinations show strong phase IIenzyme induction activity. The phase II enzyme induction assay measuresa sample's ability to induce quinone reductase (a phase II enzyme) whichis indicative of detoxification events.

Accordingly, in one embodiment, the present invention provides aformulation for improving liver health that includes wasabi root fiberpowder, artichoke leaf extract, asparagus extract, kudzu root extract,oregano extract, schisandra berry extract, notoginseng (ethanol extractof Panax notoginseng root), sanchi (water extracts from Panaxnotoginseng root), Gegen (Pueraria omeiensis), spinach dehydrate, orcombinations thereof.

In another embodiment, the present invention provides a method ofprotecting the liver from carbon tetrachloride (“CCl₄”) insults byproviding wasabi root fiber powder, artichoke leaf extract, asparagusextract, kudzu root extract, oregano extract, schisandra berry extract,notoginseng (ethanol extract of Panax notoginseng root), sanchi (waterextracts from Panax notoginseng root), Gegen (Pueraria omeiensis),spinach dehydrate, or combinations thereof.

In yet another embodiment, the present invention provides methods ofprotecting the liver from alcohol insults by providing wasabi root fiberpowder, artichoke leaf extract, asparagus extract, kudzu root extract,oregano extract, schisandra berry extract, notoginseng (ethanol extractof Panax notoginseng root), sanchi (water extracts from Panaxnotoginseng root), Gegen (Pueraria omeiensis), spinach dehydrate, orcombinations thereof.

In yet another embodiment, the present invention provides methods ofinducing phase II enzymes by providing wasabi root fiber powder,artichoke leaf extract, asparagus extract, kudzu root extract, oreganoextract, schisandra berry extract, notoginseng (ethanol extract of Panaxnotoginseng root), sanchi (water extracts from Panax notoginseng root),Gegen (Pueraria omeiensis), spinach dehydrate, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of various formulations inprotecting the liver from ethanol insults as measured by MTT.

FIG. 2 is a bar graph showing the effect of various formulations inprotecting the liver from ethanol insults as measured by LDH.

FIG. 3 is a bar graph showing the effect of various formulations inprotecting the liver from CCl₄ insults as measured by MTT.

FIG. 4 is a bar graph showing the effect of various formulations inprotecting the liver from CCl₄ insults as measured by LDH.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that this invention is not limited to theparticular methodology or protocols described herein. Further, unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood to one of ordinary skill in theart to which this invention belongs. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the claims.

Ingredients for Use in Formulations of the Present Invention

The present invention is based on the surprising discovery that uniquecombinations of the following ingredients, described more fully in Table1, improve liver health: wasabi root fiber powder, artichoke leafextract, asparagus extract, kudzu root extract, oregano extract,schisandra berry extract, notoginseng (ethanol extract of Panaxnotoginseng root), sanchi (water extracts from Panax notoginseng root),Gegen (Pueraria omeiensis), and spinach dehydrate.

TABLE 1 Ingredient Description Artichoke leaf extract Extracts fromartichoke have been used in folk medicine against (Cynara scolymus L.)liver complaints and such extracts have been claimed to exerthepatotprotective effect. Journal of Ethno-pharmacology, 86, 203-211(2003). Constituents include flavanoids and sesquiterpenoid bittercompounds of the guaianolide-type. Czygan, Franz-Christian et al.,Herbal Drugs and Phytopharmaceuticals, 3rd ed., Stuttgart, Germany,Medpharm Scientific Publishers, 2004. p. 174. Artichoke leaf extract canbe obtained from Grupo Centroflora; São Paulo, Brazil. Asparagusdehydrate Asparagus racemosus (commonly known as Shatavari) isrecommended in Ayurvedic texts for prevention and treatment of gastriculcers, dyspepsia and as a galactogogue. A. racemosus has also been usedsuccessfully by some Ayurvedic practitioners for nervous disorders,inflammation, liver diseases and certain infectious diseases. However,no scientific proof justifying aforementioned uses of root extract of A.racemosus is available so far. Indian J Med Sci., 57(9): 408-14 (2003).Animal studies have also demonstrated beneficial antioxidant effects tothe liver. Journal of Ethnopharmacology, 71, 425-435 (2003). Thechemical constituents so far reported include flavonoids,oligosaccarides, amino acids, sulphur-contianing acids, and steroidalsaponins. Id. Kudzu root extract Kudzu or Pueraria Radix is the root ofPueraria lobata which is a (Pueraria lobata) perennial leguminous vinenative to eastern Asia. The active principles of Pueraria lobata arecoumarins, isoflavonoids (puerarin, daidzin and daidzein), and sapoinins(soyasaponins, kudzusaponins). Kudzu has been used for many disorderssuch as fevers, gastrointerstinal disorders, muscle aches, allergies,respiratory problems, skin problems, high blood pressure, migraineheadaches, lowering cholesterol, and treating chronic alcoholism.Clinical Chimica Acta, 347, 121-128 (2004). Notoginseng Notoginseng asreferred to herein is the ethanol extract from (Panax notoginseng) Panaxnotoginseng root. The active constituents of Panax notoginseng includesaponins (primarily ginsenosides), dencichines, flavonoids, andpolysaccharide; however, the levels of these components vary indifferent geographical regions of growth and also show a seasonalvariation. Dong, TT et al., Chemical assessment of roots of Panaxnotoginseng in China: regional and seasonal variations in its activeconstituents. J. Agric Food Chem, 51(16): 4617-23 (2003). Panaxnotoginseng is used to treat coronary heart disease, cardiac angina, andapoplexy. Notoginseng can be obtained from EUL International HerbManufacturing, La Verne, California. Gegen root extract Commonly knownas Omei Mountain Kudzu Vine. (Pueraria omeiensis) Oregano extract Theconstituents of oregano include two phenoles carvacrol and thymol (seealso thyme and savory.) A variety of monoterpene hydrocarbons (limonene,terpinene, ocimene, caryophyllene, beta- bisabolene and p-cymene) andmonoterpene alcohols (linalool, 4- terpineol) have also been found.Sanchi Sanchi is referred to herein as the water extract from Panax(Panax notoginseng) notoginseng root. Panax notoginseng is known tocontain saponins. Sanchi can be obtained from Draco Natural Products,San Jose, California. Schisandra berry Schisandra berry or schizandraberry is commonly known as extract (Fructus Chinese Magnoliavine fruit.It is generally used to arrest schisandrae) discharges, promote fluidsecretion, tonify the kidney, and induce sedation. See U.S. Pat. No.6,455,078. Schisandra lignans have been cited to protect the liveragainst CCl₄ damage. Planta Medica, 61 (2); 134-7 (1995). Spinachdehydrate Spinach contains betaine which has been used clinically forliver disease. See US2003/0091615. Spinach dehydrate can be obtainedfrom Access Business Group LLC, Ada, Michigan. Wasabi root Studiessuggest that wasabi (Wasabia japonica, syn. Eutrema Wasabi) may generatean increase in the abundance of the protective phase II detoxificationenzymes, such as glutathione S- transferase (GST), resulting inhepatoprotection. Recent identification of 6-methylsulfinylhexylisothiocyanate (6-HITC), an analogue of sulforaphane(4-methylsulfinylbutyl isothiocyanate) isolated from broccoli, has beensuggested to be the major GST inducer in wasabi. J. Biol Chem., 1;277(5): 3456-63 (2002). It is known that wasabi contains isothiocyanatecomponents. Id. Wasabi root extract can be obtained from EULInternational Herb Manufacturing, La Verne, California.

More specifically, the formulations of the present invention improveliver health by protecting the liver against alcohol and carbontetrachloride insults. Additionally, the formulations improve liverhealth by inducing phase II enzymes. Phase II enzymes are responsiblefor removing potential carcinogens by aiding in their removal from thebody.

“Protecting the liver against alcohol insults” and “protecting the liveragainst carbon tetrachloride insults” refers to the ability of aformulation described herein to preserve or improve existing liverfunction.

Formulations of the Present Invention

Table 2 illustrates representative daily amounts of the ingredients thatcan be included in the supplement.

TABLE 2 Ingredient Daily Dosage Artichoke leaf extract 200 mg-1000 mgAsparagus extract 100 mg-500 mg Gegen root extract 150 mg-1000 mg Kudzuroot extract 150 mg-1000 mg Notoginseng 100 mg-500 mg Oregano extract 20 mg-500 mg Sanchi 100 mg-500 mg Schisandra berry extract 150 mg-1000mg Spinach dehydrate  20 mg-500 mg Wasabi root powder 150 mg-500 mg

EXAMPLES

The following are illustrative examples of formulations made intotablets according to this invention and it should be understood thatthey do not limit the scope of the invention.

Example 1

Per: 3 Tablets Qty. U/M Item # Ingredients % W/W 350.0 mg R7462 Sanchiroot concentrate 18.88% 300.00 mg R6911 Asparagus dehydrate 16.18%150.00 mg R7464 Gegen root extract, 10% 8.09% 500.00 mg R7461 Artichokeleaf extract, 5% 26.97% 150.00 mg NF9824Z Spinach dehydrate 8.09% 310.0mg R4174Q Microcrystalline Cellulose, 16.72% Silicified 44.0 mg R3338Cellulose Gum 2.37% 5.6 mg R0225 Silicon Dioxide, Fine Powder 0.30% 44.0Mg R3512 Stearic Acid, Powder, Vegetable 2.37% 11.0 mg NF6004Hydroxypropyl Methylcellulose

Example 2

Per: 3 Tablets Qty. U/M Item # Ingredients % W/W 500.0 mg R7461Artichoke powder extract 30.10% 150.00 mg R7460 Wasabi root concentrate9.03% 500.00 mg R7464 Kudzu root, 40% 30.10% 150.00 mg R6910 OreganoExtract 9.03% 278.0 mg R4174Q Microcrystalline Cellulose, Silicified16.74% 39.0 mg R3338 Cellulose Gum 2.35% 5.0 mg R0225 Silicon Dioxide,Fine Powder 0.30% 39.0 mg R3512 Stearic Acid, Powder, Vegetable 2.35%11.0 mg HPMC Hydroxypropyl Methylcellulose

Example 3

Per: 3 Tablets Qty. U/M Item # Ingredients % W/W 150.00 mg R7462 Sanchi8.15% 150.00 mg R7521 Notoginseng 8.15% 500.00 mg R7461 Artichoke leafextract, 5% 27.18% 500.00 mg R7463 Kudzu root extract 27.18% 150.00 mgR6910 Oregano Extract 8.15% 300.0 mg R4174Q Microcrystalline Cellulose,Silicified 16.31% 42.0 mg R3338 Cellulose Gum, Modified NF 2.28% 5.5 mgR0225 Silicon Dioxide, NF Fine Powder 0.30% 42.0 mg R3512 Stearic Acid,Powder, Vegetable 2.28% 13.0 mg NF6004 Hydroxypropyl Methylcellulose

Example 4

Per: 3 Tablets Qty. U/M Item # Ingredients % W/W 350.0 mg R7462 Sanchi20.42% 300.00 mg R6911 Asparagus dehydrate 17.50% 500.00 mg R7461Artichoke leaf extract, 5% 29.17% 200.00 mg NF9824Z Spinach dehydrate11.67% 280.0 mg R4174Q Microcrystalline Cellulose, 16.34% Silicified39.0 mg R3338 Cellulose Gum 2.28% 5.0 mg R0225 Silicon Dioxide, FinePowder 0.29% 40.0 mg R3512 Stearic Acid, Powder, Vegetable 2.33% 12.0 mgNF6004 Hydroxypropyl Methylcellulose

Example 5

Per: 3 Tablets Qty. U/M Item # Ingredients % W/W 500.00 mg R7461Artichoke leaf extract 27.18% 500.00 mg R7463 Kudzu root extract, 40%27.18% 150.00 mg R7521 Notoginseng 8.15% 150.0 mg R7460 Wasabi fiberpowder 8.15% 150.00 mg NF9824Z Spinach dehydrate 8.15% 300.0 mg R4174QMicrocrystalline Cellulose, 16.31% Silicified 42.0 mg R3338 CelluloseGum 2.28% 5.6 mg R0225 Silicon Dioxide, Fine Powder 0.30% 42.0 mg R3512Stearic Acid, Powder, Vegetable 2.28% 11.0 mg NF6004 HydroxypropylMethylcellulose

Example 6

Per 3 Tablets Qty. U/M Item # Ingredients % W/W 500.0 mg R7461 Artichokeleaf extract, 5% 23.88% 500.00 mg R7460 Wasabi fiber Powder 23.88%500.00 mg R7464 Gegen Root extract, 10% 23.88% 150.00 mg R6910 OreganoExtract 7.16% 342.0 mg R4174Q Microcrystalline Cellulose, Silicified16.33% 48.0 mg R3338 Cellulose Gum 2.29% 6.2 mg R0225 Silicon Dioxide,Fine Powder 0.30% 48.0 mg R3512 Stearic Acid, Powder, Vegetable 2.29%11.0 mg NF6004 Hydroxypropyl Methylcellulose

Example 7

Per 2 Tablets Qty. U/M Item # Ingredients % W/W 150.0 mg R7460 Wasabiroot fiber powder 8.83% 500.00 mg R7461 Artichoke leaf extract, 5%29.43% 300.00 mg R3490 Schizandra berry ext 17.66% 150.00 mg R7521Notoginseng 8.83% 150.00 mg NF9824Z Spinach dehydrate 8.83% 346.0 mgR4174Q Microcrystalline Cellulose, 20.36% Silicified 49.0 mg R3338Cellulose Gum 2.88% 5.1 mg R0225 Silicon Dioxide, Fine Powder 0.30% 49.0mg R3512 Stearic Acid, Powder, Vegetable 2.88% 11.0 mg NF6004Hydroxypropyl Methylcellulose

Example 8

Per 3 Tablets Qty. U/M Item # Ingredients % W/W 150.0 mg R7460 Wasabiroot fiber powder 7.62% 500.00 mg R7461 Artichoke leaf extract, 5%25.41% 500.00 mg R7463 Kudzu root, 40% 25.41% 150.00 mg R7521Notoginseng 7.62% 150.00 mg R6910 Oregano extract 7.62% 400.0 mg R4174QMicrocrystalline Cellulose, Silicified 20.33% 55.0 mg R3338 CelluloseGum 2.79% 5.9 mg R0225 Silicon Dioxide, Fine Powder 0.30% 38.0 mg R3512Stearic Acid, Powder, Vegetable 2.90% 11.0 mg NF6004 HPMC

Example 9

Label Per: 2 Tablets Qty. U/M Item # Ingredients % W/W 500.00 Mg R7461Artichoke leaf extract, 5% 32.16% 500.00 Mg R7463 Kudzu root extract,40% 32.16% 150.00 Mg R6910 Oregano Extract 9.65% 315.0 mg R4174QMicrocrystalline Cellulose, Silicified 20.26% 40.0 Mg R3338 CelluloseGum, Modified NF 2.57% 4.7 mg R0225 Silicon Dioxide, NF Fine Powder0.30% 45.0 mg R3512 Stearic Acid, Powder, Vegetable 2.89% 11.0 mg NF6004HPMC

The above exemplary tabletted formulations can be manufactured accordingto typical methods known in the industry. For example, wasabi root fiberpowder, artichoke leaf extract, schizandra berry extract, notoginsengand spinach dehydrate are passed through a SWECO separator equipped witha 20 mesh screen into a 100 cubic foot PK blender. Microcrystallinecellulose is added to the blend in the PK blender. The ingredients areblended for ten minutes. Cellulose gum and silicon dioxide are passedthrough a SWECO separator equipped with a 20 mesh screen directly intothe 100 cubic foot PK blender. The ingredients are blended for tenminutes. Next, stearic acid is passed through a SWECO separator equippedwith a 20 mesh screen directly into the 100 cubic foot PK blender. Themixture is blended for an additional five minutes. The resulting mixtureis discharged into totes or supersacks, and compressed into tablets.

Methods of Administration

Formulations of the present invention may be formulated in an acceptablecarrier and may be prepared, packaged, and labeled for promoting health,liver function, protecting against alcohol and/or chemical insults tothe liver, and/or inducing phase II enzymes to promote healthy liverfunction. The formulations of the present invention and their acceptablecarriers may be formulated for oral administration in the form of apill, tablet, dried or powdered product for reconstitution with water orother suitable vehicle before use, bar, food, solution, syrup,suspension, beverage, lozenge, etc. The formulations of the presentinvention may also be parenterally administered or administered byinhalation or insufflation (either through the mouth or nose).

Liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). When administered in the form of a beverage, formulations of thepresent invention may be water-based, milk-based, tea-based, fruitjuice-based, or some combination thereof.

Formulations of the present invention may also be orally administered inthe form of a solid prepared by conventional means with pharmaceuticallyacceptable excipients such as binding agents (e.g., pregelatinized maizestarch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose(“HPMC”)); fillers (e.g., lactose, microcrystalline cellulose or calciumhydrogen phosphate); lubricants (e.g., magnesium stearate, talc orsilica); disintegrants (e.g., cellulose gum, potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate).

Formulations of the present invention that are orally administered canfurther comprise thickeners, including xanthum gum,carbosymethyl-cellulose, carboxyethylcellulose, hydroxyporpolcellulose,methylcellulose, microcrystalline cellulose, starches, dextrins,fermented whey, tofu, maltodextrins, polyols, including sugar alcohols(e.g., sorbitol and mannitol), carbohydrates (e.g. lactose), propyleneglycol alginate, gellan gum, guar, pectin, tragacanth gum, gum acacia,locust bean gum, gum arabic, gelatin, as well as mixtures of thesethickeners.

Orally administered formulations of the present invention can contain aneffective amount of one or more sweeteners, including carbohydratesweeteners and natural and/or artificial no/low calorie sweeteners. Theamount of the sweetener used in the formulations of the presentinvention will vary, but typically depends on the type of sweetener usedand the sweetness intensity desired.

In addition to the formulations described previously, the compounds mayalso be a formulated as a sustained and/or timed release formulation.The formulations must be maintained above some minimum therapeutic doseto be effective. Common timed and/or controlled release delivery systemsinclude, but are not be restricted to, starches, osmotic pumps, orgelatin micro capsules.

The formulations may, if desired, be presented in a pack or dispenserdevice which may comprise one or more unit dosage forms comprising aformulation of the present invention. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

Other useful dosage forms can be prepared by methods and techniques thatwill be well understood by those of skill in the art and may include theuse of additional ingredients in producing tablets, capsules, or liquiddosage forms. The dose, and dose frequency, will vary according to theage, body weight, condition and response of the individual consumer orpatient, and the particular formulation of the present invention that isused.

It is intended that the foregoing detailed description be regarded asillustrative rather than limiting. The present invention is furtherillustrated by the following experimental investigations and examples,which should not be construed as limiting.

Bioassay Study on Individual Ingredients

Several materials were submitted for bioassay testing to try to predicttheir protective ability against alcohol and chemically induced liverdamage. To assess this, human liver cells were treated with the materialand an insult, and cell viability was measured using two differentassays. Alcohol damage was mimicked using 2.5% ethanol as the insult.CCl₄ at 0.2% was used for the chemical insult. These concentrations werechosen because they were the concentrations that produced 20% cell deathin preliminary experiments, and would therefore not cause the cells toundergo irreversible cell death/damage.

Materials were tested at three concentrations (1, 10 and 100 μg/mL) andan estimation of the EC-50 was determined (see Experimental sectionbelow for more details). In addition to the cell viability assays, PhaseII Enzyme Induction testing was also conducted to determine if amaterial had detoxification properties. The Phase II Enzyme Inductionassay measures a sample's ability to induce quinone reductase (a PhaseII enzyme) which is indicative of detoxification events. Phase IIenzymes are responsible for removing potential carcinogens by aiding intheir removal from the body. Broccoli is known as a good Phase II enzymeinducer because of its sulforaphane content. Pure sulforaphane hasactivity at 10⁶ U/g, while broccoli has reported activity from4000-74,000 U/g (dependent on variety, form, and/or extractionconditions). Activity above 30,000 U/g is considered excellent. Activityless than 5000 U/g is considered minimal. Most materials that areconsidered good Phase II Enzyme inducers will have activity between15,000-30,000 U/g.

Tables 3 and 4 summarize the EC-50 values of all materials tested. Table3 is specifically for results of the ethanol protection, and Table 4 forcarbon tetrachloride. Table 5 gives the results of the Phase II EnzymeInduction assay.

TABLE 3 EC-50 results of the MTT and LDH assays for Ethanol protectionassessment. EC-50 EC-50 MTT LDH Sample Description (μg/mL) (μg/mL)CD-2808 Hibiscus SD Powder 10-100 None CD-4230 Picao Pret (BidensPilosa) Herb None >100 CD-4228 Pueraria (Kudzu) Root 40% <1 None CD-3902Cardoon SD Powder >100 None CD-4229 Gegen root extract (pueraria NoneNone omeiensis), 10% CD-4796 Beet Powder 100 >100 CD-4775 Aframomum S/DPowder None >100 CD-4788 Schisandra berry Extract None >100 CD-4780Asparagus Dehydrate None None CD-4793 Bidens Pilosa Extract None NoneCD-4787 Picrorhiza Kurrooa Extract None >100 CD-4786 Eclipta AlbaExtract None None CD-4795 Curcumin 95% None None CD-4792 Boldo ExtractNone >100 CD-5347 Szechuan Lovage Rhizome <1 >100 CD-5345 Sanchi <1 >100CD-5346 Glycyrrhiza Uralensis Fisch Root <1 >100 CD-5348 ArtichokeExtract Powder, 5% <1 >100 Cynarin CD-5349 Artichoke Extract Powder,2.5% <1 >100 Cynarin CD-5350 Chanca Piedra Extract 3:1 <1 >100 CD-5383Kudzu Extract Powder 40% None None CD-5384 Tree Peony Bark 1:8 None NoneCD-5385 Gegen Extract None None CD-5386 Pueraria 40% <1 >100 CD-5442Curcuma Longa Extract 10:1 None None CD-5441 Moutan Extract 12:1 NoneNone CD-5440 Notoginseng Extract 10% None 10-100 CD-5548 Silybin ComplexNone None CD-5550 Dandelion 4:1 None None CD-5549 Milk Thistle 70%Granular None None CD-5624 Oregano S/D Powder None None CD-5728 HolyBasil S/D Powder None None CD5730 Holy Basil S/D Powder None NoneCD-5741 Sage S/D Powder None None CD-5241 Gegen Extract 10% None 10-100CD-5796 Radix Notoginseng None 10-100 CD-5797 Artichoke Extract Powder5% 1 >100 CD-5798 Rhizoma Curcumae Longae None None CD-5799 RhizomaChuanxiong None >100 CD-5800 Semen Armeniacae Amarum None >100 CD-5801Tree Bark Extract 8:1 None >100 CD-5802 Cortex Moutan None None CD-5803Radix Glycyrrhizae >100 >100 CD-5707 Wasibia Japonica Powder 100%None >100 CD-5863 Wasabi Root P.E. 5:1 >100   100 CD-5864 Wasabi EarhnutP.E. 5:1 10-100 10-100 CD-5865 Wasabi Fibre P.E. 5:1 10-100 >100 CD-5866Wasabi Root Powder >100 >100 CD-5867 Wasabi Earthnut Powder >100 >100CD-5868 Wasabi Fibre Powder >100 >100

TABLE 4 EC-50 results of the MTT and LDH assays for CCl4 protectionassessment. EC-50 EC-50 MTT LDH Sample Description (μg/mL) (μg/mL)CD-2808 Hibiscus SD Powder None 1 CD-4230 Picao Pret (Bidens Pilosa)Herb 1-10 None CD-4228 Pueraria (Kudzu) Root 40% 1-10 None CD-3902Cardoon SD Powder None <1 CD-4229 Gegen (Kudzu Root) 10% None <1 CD-4796Beet Powder None 1 CD-4775 Aframomum S/D Powder None <1 CD-4788Schisandra Berry Extract None 1-10 CD-4780 Asparagus Dehydrate None <1CD-4793 Bidens Pilosa Extract None <1 CD-4787 Picrorhiza Kurrooa Extract100 1 CD-4786 Eclipta Alba Extract >100 <1 CD-4795 Curcumin 95% None <1CD-4792 Boldo Extract None <1 CD-5347 Szechuan Lovage Rhizome >100 NoneCD-5345 Sanchi 10-100 1-10 CD-5346 Glycyrrhiza Uralensis Fisch Root >1001-10 CD-5348 Artichoke Extract Powder, 5% 100 <1 Cynarin. CD-5349Artichoke Extract Powder, 2.5% >100 None Cynarin CD-5350 Chanca PiedraExtract 3:1 >100 100 CD-5383 Kudzu Extract Powder 40% 10-100 100 CD-5384Tree Peony Bark 1:8 10-100 1-10 CD-5385 Gegen Extract 10-100 <1 CD-5442Curcuma Longa Extract 10:1 <1 1-10 CD-5441 Moutan Extract 12:1 1-10 1-10CD-5440 Notoginseng Extract 10% <1 <1 CD-5548 Silybin Complex None NoneCD-5550 Dandelion 4:1 10-100 >100 CD-5549 Milk Thistle 70% GranularNone >100 CD-5624 Oregano S/D Powder None >100 CD-5728 Holy Basil S/DPowder None >100 CD5730 Holy Basil S/D Powder None None CD-5741 Sage S/DPowder None None CD-5241 Gegen Extract 10% 10-100 1-10 CD-5796 RadixNotoginseng 1-10 >100 CD-5797 Artichoke Extract Powder 5% 1-10 1-10CD-5798 Rhizoma Curcumae Longae None 1-10 CD-5799 Rhizoma ChuanxiongNone >100 CD-5800 Semen Armeniacae Amarum None None CD-5801 Tree BarkExtract 8:1 >100 >100 CD-5802 Cortex Moutan 10-100 None CD-5803 RadixGlycyrrhizae 10-100 None CD-5707 Wasibia Japonica Powder 100% >100 >100CD-5863 Wasabi Root P.E. 5:1 >100 1-10 CD-5864 Wasabi Earhnut P.E. 5:110-100 10-100 CD-5865 Wasabi Fibre P.E. 5:1 10 1-10 CD-5866 Wasabi RootPowder None 1-10 CD-5867 Wasabi Earthnut Powder 1-10 10-100 CD-5868Wasabi Fibre Powder 10-100 10-100

TABLE 5 Phase II Enzyme (Quinone Reductase) Induction Assay ResultsSample Description U/g Rank CD-2808 Hibiscus SD Powder 14,493 ++ CD-4230Picao Pret (Bidens Pilosa) Herb 24,691 ++ CD-4228 Kudzu Root 40% 25,641++ CD-3902 Cardoon SD Powder Not Tested Not Tested CD-4229 Gegen (KudzuRoot) 10% 95,238 ++++ CD-4796 Beet Powder 9132 ++ CD-4775 Aframomum S/DPowder Not Tested Not Tested CD-4788 Schisandra Berry Extract 30,303++++ CD-4780 Asparagus Dehydrate Not Tested Not Tested CD-4793 BidensPilosa Extract 9132 ++ CD-4787 Picrorhiza Kurrooa Extract 5420 ++CD-4786 Eclipta Alba Extract 10,101 ++ CD-4795 Curcumin 95% NR** NR**CD-4792 Boldo Extract 13,605 ++ CD-5347 Szechuan Lovage Rhizome 11,299++ CD-5345 Sanchi 13,333 ++ CD-5346 Glycyrrhiza Uralensis Fisch 14,815++ Root CD-5348 Artichoke Extract Powder, 5% 27,778 +++ Cynarin CD-5349Artichoke Extract Powder, 2.5% 44,444 ++++ Cynarin CD-5350 Chanca PiedraExtract 3:1 95,238 ++++ CD-5383 Kudzu Extract Powder 40% 55,556 ++++CD-5384 Tree Peony Bark 1:8 11,111 ++ CD-5385 Gegen Extract 95,238 ++CD-5442 Curcuma Longa Extract 10:1 NR* NR* CD-5441 Moutan Extract 12:111,111 ++ CD-5440 Notoginseng Extract 10% 6006 ++ CD-5548 SilybinComplex NR** NR** CD-5550 Dandelion 4:1 NR** NR** CD-5549 Milk Thistle70% Granular NR** NR** CD-5624 Oregano S/D Powder 95,238 ++++ CD-5728Holy Basil S/D Powder 31,746 ++++ CD5730 Holy Basil S/D Powder 31,746++++ CD-5741 Sage S/D Powder 35,088 ++++ CD-5241 Gegen Extract 10%37,037 ++++ CD-5796 Radix Notoginseng 15,504 +++ CD-5797 ArtichokeExtract Powder 5% 9524 ++ CD-5798 Rhizoma Curcumae Longae 83,333 ++++CD-5799 Rhizoma Chuanxiong 27,778 +++ CD-5800 Semen Armeniacae Amarum22,222 +++ CD-5801 Tree Bark Extract 8:1 17,544 +++ CD-5802 CortexMoutan 21,505 +++ CD-5803 Radix Glycyrrhizae 11,494 ++ CD-5707 WasibiaJaponica Powder 100% 74,074 ++++ CD-5863 Wasabi Root P.E. 5:1 10,753 ++CD-5864 Wasabi Earhnut P.E. 5:1 83,333 ++++ CD-5865 Wasabi Fibre P.E.5:1 12,346 ++ CD-5866 Wasabi Root Powder 30,303 ++++ CD-5867 WasabiEarthnut Powder 5012 ++ CD-5868 Wasabi Fibre Powder >133,333 ++++CD-5914 Spinach Dehydratw 37,037 ++++ *Inducer units per gram of freshweight of material. − = Negligible activity, + = Little activity, ++ =Good activity, +++ = Very good activity, ++++ = Excellent activity.**Not reportable due to toxicity to cells.Experimental

For the ethanol protection and CCl₄ assessments, stock sample solutionsare made in DMSO, then diluted in cell culture media for testing.Treatment of HepG2 cells (human liver cell line) is done by adding 100μL of sample to each of three wells of a 96-well microtiter plate. Aftera 4 hour incubation, the insult is added (2.5% ethanol or 0.2% carbontetrachloride) and an additional overnight incubation period isconducted. The next day, cell viability is measured using two differentassays. First, using the CytoTox-ONE Homogenous Membrane Integrity Assayby Promega, the number of non-viable cells is estimated by measuring therelease of lactate dehydrogenase (LDH) into the media. LDH leaks out ofthe cell when the cell membrane is compromised. The second assay is theMTT assay, which measures the reduction of a yellow tetrazolim salt(MTT) into an insoluble purple formazen product by the mitochondria ofviable cells. Following an incubation with the MTT solution, isopropanolis added to solubilize the colored crystals. The amount of colorproduced is directly proportional to the number of viable cells.

For the phase II enzyme induction assay, stock sample solutions are madein acetonitrile, then diluted in cell culture media for testing.Treatment of Hepa1c1c7 cells (murine hepatoma cell line) is done byadding 150 μL of sample to each of three wells, in a 96-well microtiterplate. After 48 hours incubation, induction activity of quinonereductase is established by measuring the NADPH-dependent,menadiol-mediated reduction of MTT. Activity is reported as inducerunits per gram of fresh weight of material, where one unit of induceractivity is defined as the amount of inducer required to double thequinone reductase specific activity of Hepa1c1c7 cells.

Bioassay Study on Ingredient Combinations

Several blends of liver detoxification ingredients were submitted forbioassay testing to try to predict their protective ability againstalcohol and chemically induced liver damage. These blends were testedalongside two products already on the market for liver health—NUTRILITE®Milk Thistle and Dandelion and China's King Drink. To assess this, humanliver cells were treated with the sample and an insult, and cellviability was measured using two different assays. Alcohol damage wasmimicked using 2.5% ethanol as the insult. CCl₄ at 0.2% was used for thechemical insult. These concentrations were chosen because they were theconcentrations that produced 20% cell death in preliminary experiments,and would therefore not cause the cells to undergo irreversible celldeath/damage. Samples were tested at three concentrations (1, 10 and 100μg/mL) and an estimation of the EC-50 was determined (see Experimentalsection below for more details). Materials with EC-50 values at or below10 μg/mL are indicative of being the most efficacious, assuming 10%absorption of the material in 5 L of blood (average human volume).

The blends that showed the greatest efficacy against CCl₄ liver celldamage were 8523-25-CI (Example 1), 8523-27-CI (Example 8), 8523-28-CI(Example 2), and 8523-30-CI (Example 3). Other blends that performedwell (exhibited 40% protection at ≦10 μg/mL) were 8523-20-CI,8523-22-CI, 8523-24-CI, 8523-26-CI and 8523-31-CI. None of the blendsshowed efficacy at ≦10 μg/mL against ethanol liver cell damage. Thecontrol products (NUTRILITE milk thistle and King Drink) also did notachieve EC-50 values of ≦10 μg/mL. The blends that had EC-50 values >100μg/mL were 8523-28-CI (Example 2), 8523-30-CI (Example 3), 8523-31-CI(Example 4) and 8523-32-CI (Example 5). Tables 6-9 summarize the resultsof all the samples tested. These results are also seen in FIGS. 1-4.

TABLE 6 Results of the MTT assays for ethanol protection assessmentFormula Sample Number or Number Sample Name 1 ug/mL +/− 10 ug/mL +/− 100ug/mL +/− EC-50 NF6523 8523-20-CI −73% 6% −58% 4% −1% 7% — NF65238523-21-CI −80% 5% −82% 3% −97% 11% — NF6523 8523-22-CI 43% 4% −4% 4%−52% 12% * NF6523 8523-23-CI −63% 2% −55% 4% −44% 3% — NF6523 8523-24-CI−61% 1% −72% 2% −70% 4% — NF6523 8523-25-CI −42% 4% 31% 3% −7% 1% —NF6523 8523-26-CI −51% 6% −54% 5% −49% 11% — NF6523 8523-27-CI 6% 6%−58% 4% −53% 6% — NF6523 8523-28-CI 22% 22% 12% 18% 41% 15% >100 NF65238523-30-CI 9% 25% −48% 17% −43% 16% — NF6523 8523-31-CI −65% 3% −39% 4%−22% 5% — NF6523 8523-32-CI −67% 8% −77% 8% −88% 5% — NF6523 8523-33-CI19% 8% −35% 20% −62% 8% — Milk Thistle −53% 3% −54% 8% −42% 4 — CD5439King Drink −57% 3% −13% 17% −8% 6% — (*= achieved EC-40 at one or moreconcentration)

TABLE 7 EC-50 results of the LDH assays for ethanol protectionassessment. Formula Sample Number or Number Sample Name 1 ug/mL +/− 10ug/mL +/− 100 ug/mL +/− EC-50 NF6523 8523-20-CI −60% 20% −39% 12% −33%5% — NF6523 8523-21-CI −53% 17% −51% 15% −77% 8% — NF6523 8523-22-CI−27% 4% −6% 16% −55% 11% — NF6523 8523-23-CI −82% 7% −77% 4% −80% 4% —NF6523 8523-24-CI −69% 2% −68% 9% −61% 5% — NF6523 8523-25-CI −62% 8%−19% 2% −25% 4% — NF6523 8523-26-CI −65% 7% −94% 7% −76% 1% — NF65238523-27-CI −17% 6% −75% 6% −54% 6% — NF6523 8523-28-CI −35% 15% −2% 5%−8% 13% — NF6523 8523-30-CI −62% 10% −47% 14% −43% 20% — NF65238523-31-CI 8% 18% 12% 5% 6% 23% >100 NF6523 8523-32-CI 14% 23% 10% 21%7% 25% >100 NF6523 8523-33-CI 5% 2% 25% 1% 13% 24% >100 Milk Thistle−23% 2% −24% 6% −7% 7% — CD5439 King Drink −26% 10% 1% 6% 1% 12% >100(*= achieved EC-40 at one or more concentration)

TABLE 8 EC-50 results of the MTT assays for CCl4 protection assessment.Formula Sample Number or Number Sample Name 1 ug/mL +/− 10 ug/mL +/− 100ug/mL +/− EC-50 NF6523 8523-20-CI −42% 10% −60% 11% −6% 17% — NF65238523-21-CI −95% 15% −78% 6% −66% 17% — NF6523 8523-22-CI −47% 22% −120%21% −97% 3% — NF6523 8523-23-CI −56% 12% −57% 15% −58% 17% — NF65238523-24-CI −12% 12% −80% 18% −50% 14% — NF6523 8523-25-CI 42% 1% 58% 2%9% 29% 1-10 NF6523 8523-26-CI −24% 4% −13% 11% −38% 22% — NF65238523-27-CI 8% 13% −9% 15% −61% 5% — NF6523 8523-28-CI −66% 8% 58% 7%−17% 17% 1-10 NF6523 8523-30-CI −84% 19% 54% 11% −59% 23% 1-10 NF65238523-31-CI −33% 7% −45% 16% 23% 7% >100 NF6523 8523-32-CI −91% 10% −39%5% 4% 16% >100 NF6523 8523-33-CI −19% 8% −17% 12% −25% 18% — MilkThistle −18% 12% −54% 1% −42% 8% — CD5439 King Drink −67% 13% 33% 6%−22% 13% — (*= achieved EC-40 at one or more concentration)

TABLE 9 EC-50 results of the LDH assays for CCl4 protection assessment.Formula Sample Number or Number Sample Name 1 ug/mL +/− 10 ug/mL +/− 100ug/mL +/− EC-50 NF6523 8523-20-CI 44% 11% 35% 5% 25% 14% * NF65238523-21-CI 20% 20% 35% 11% 22% 15% — NF6523 8523-22-CI 22% 8% 42% 27%34% 2% * NF6523 8523-23-CI 36% 13% 38% 5% 26% 6% — NF6523 8523-24-CI 42%2% 39% 26% 39% 3% * NF6523 8523-25-CI 38% 9% 23% 1% 47% 11% 100 NF65238523-26-CI 34% 6% 40% 9% 36% 14% * NF6523 8523-27-CI 47% 0% 44% 0% 42%9% 1 NF6523 8523-28-CI 37% 10% 48% 20% 30% 5% 10 NF6523 8523-30-CI 32%6% 39% 2% 40% 4% * NF6523 8523-31-CI 43% 11% 31% 2% 22% 6% * NF65238523-32-CI −1% 27% 3% 28% 25% 26% >100 NF6523 8523-33-CI 15% 18% −27%28% −20% 28% — Milk Thistle 31% 17% 29% 1% 26% 18% >100 CD5439 KingDrink 30% 13% 32% 6% 50% 19% 100 (*= achieved EC-40 at one or moreconcentration)

In addition to the cell viability assays, phase II enzyme inductiontesting was also conducted to determine if a material had detoxificationproperties. The phase II enzyme induction assay measures a sample'sability to induce quinone reductase (a phase II enzyme) which isindicative of detoxification events. Broccoli is known as a good phaseII enzyme inducer because of its sulforaphane content. Pure sulforaphanehas activity at 10⁶ U/g, while broccoli has reported activity from4000-74,000 U/g (dependent on variety, form, and/or extractionconditions). Activity above 30,000 U/g is considered excellent. Activityless than 5,000 U/g is considered minimal. Most materials that areconsidered good phase II enzyme inducers will have activity between15,000-30,000 U/g.

All the blends tested had good to excellent phase II enzyme inductionactivity. The highest activity came from 8523-27 (Example 8) and thelowest from 8523-31 (Example 4). Excellent activity also came from8523-22, (Example 7) 8523-23, 8523-30 (Example 3), 8523-32 (Example 5)and 8523-33. Table 10 gives the results of the Phase II Enzyme Inductionassay for all samples tested.

TABLE 10 Phase II Enzyme (Quinone Reductase) Induction Assay ResultsSample Formula Number or Number Sample Name U/g Rank NF6523 8523-20-CI23,810 +++ NF6523 8523-21-CI 24,690 +++ NF6523 8523-22-CI 51,280 ++++NF6523 8523-23-CI 31,750 ++++ NF6523 8523-24-CI 15,500 +++ NF65238523-25-CI 26,670 +++ NF6523 8523-26-CI 16,260 +++ NF6523 8523-27-CI83,330 ++++ NF6523 8523-28-CI 19,610 +++ NF6523 8523-30-CI 44,440 ++++NF6523 8523-31-CI 9950 ++ NF6523 8523-32-CI 47,620 ++++ NF65238523-33-CI 60,600 ++++ Milk Thistle >100,000 ++++ CD5439 King Drink17,540 +++ *Inducer units per gram of fresh weight of material. − =Negligible activity, + = Little activity, ++ = Good activity, +++ = Verygood activity, ++++ = Excellent activity. **Not reportable due totoxicity to cells.Experimental

Stock sample solutions are made in DMSO, then diluted in cell culturemedia for testing. Treatment of HepG2 cells (human liver cell line) isdone by adding 100 μL of sample to each of three wells of a 96-wellmicrotiter plate. After a 4 hour incubation, the insult is added (2.5%ethanol or 0.2% carbon tetrachloride) and an additional overnightincubation period is conducted. The next day, cell viability is measuredusing two different assays. First, using the CYTOTOX-ONE™ HomogenousMembrane Integrity Assay by Promega, the number of non-viable cells isestimated by measuring the release of lactate dehydrogenase (“LDH”) intothe media. LDH leaks out of the cell when the cell membrane iscompromised. The second assay is media. LDH leaks out of the cell whenthe cell membrane is compromised. The second assay is the MTT assay,which measures the reduction of a yellow tetrazolim salt (“MTT”) into aninsoluble purple formazen product by the mitochondria of viable cells.Following an incubation with the MTT solution, isopropanol is added tosolubilize the colored crystals. The amount of color produced isdirectly proportional to the number of viable cells.

Protection is determined by first calculating a percent toxicity of eachwell (1—experimental/negative control), after averaging the threereplicates. Percent protection is then calculated by the following: (%Toxicity Positive Control−% Toxicity Sample)/% Toxicity PositiveControl, the positive control being either 2.5% ethanol or 0.2% carbontetrachloride. The concentration that exhibits 50% protection (EC-50)can then be assessed. For the purpose of this experiment, this wascategorized as either <1, 1, 1-10, 10, 10-100, 100 or >100 μg/mL.

For the Phase II Enzyme Induction Assay, stock sample solutions are madein acetonitrile, then diluted in cell culture media for testing.Treatment of Hepa1c1c7 cells (murine hepatoma cell line) is done byadding 150 μL of sample to each of three wells, in a 96-well microtiterplate. After 48 hours incubation, induction activity of quinonereductase is established by measuring the NADPH-dependent,menadiol-mediated reduction of MTT. Activity is reported as inducerunits per gram of fresh weight of material, where one unit of induceractivity is defined as the amount of inducer required to double thequinone reductase specific activity of Hepa1c1c7 cells.

Mammalian Studies

Clinical testing can be conducted to confirm the efficacy of theformulations on liver health. It is expected that the formulations willimprove liver health by protecting the liver from chemical and alcoholinsults. A protocol for such testing follows.

Protocol 1: CCl₄ Liver Injury Model

1.1 Principles.

When CCl₄ is activated by microsomal enzyme of liver, trichloromethanefree radicals (CCl₃.) are formed. Covalent combination of this freeradical with protein results in impairment of protein synthesis anddisorder of lipid catabolism, causing accumulation of triglyceride (TG)in liver cells. CCl₃. also can combine with O₂ rapidly to formtrichloromethane peroxide free radicals (CCl₃O₂.), leading to lipidperoxidation which causes degenerative injury of cell membrane, leakageof enzymes and various types of pathological changes of cells and evennecrosis.

1.2 Experimental Animals.

Adult rats or mice of single sex. Each group consists of 8-12 rats(180-220 g) or 10-15 mice (18-22 g).

1.3 Experimental Methods and Procedures.

1.3.1 Dosage Groups and Duration of Administration of the Test Sample.

Three dosage groups, one blank control group and one model control groupare set. The dosage of one of the dosage groups is 10 times (mice) or 5times (rats) the recommended human dosage. CCl₄ (analytically pure) isused to form liver injury model. The method of forming model can useintragastric administration or intraperitoneal injection. Theconcentration of CCl₄ for intragastric administration in mice is 1%.CCl₄ is diluted with edible vegetable oil and the dosage forintragastric administration is 5 mL/kg BW (the dosage in terms of CCl₄is 80 mg/kg BW). The concentration of CCl₄ for intragastricadministration in rats is 2%-3% and the dosage is 5 mL/kg BW (the dosagein terms of CCl₄ is 160-240 mg/kg BW). Positive control group andsolvent control group may be set if necessary. The duration ofadministration of the test sample is 30 days and can be prolonged to 45days if necessary.

1.3.2 Route of Administration of the Test Sample.

The test sample is given intragastrically. If this is impossible, thetest sample can be mixed into the feed or drinking water and the feedintake or water drunk is recorded.

1.3.3 Experimental Procedures.

The animals of the experimental group are given intragastrically thetest sample daily, while those of the blank control group and the modelcontrol group are given distilled water. The animals are weighed twice aweek for adjusting the dosage of the test sample. On the eve of day 30of the experiment, the animals of various groups fast for 16 h. Theanimals of the model group and various test sample groups are givenintragastrically single dose of CCl₄, while those of the blank controlgroup are given vegetable oil. The animals of the experimental groupcontinue to receive the test sample until the end of the experiment (theinterval between administration of the test sample and CCl₄ is over 4h). After giving CCl₄, the animals are sacrificed 24 h or 48 h lateraccording to the actual conditions. Blood is taken and serum isseparated for measuring ALT and AST. Liver is taken forhistopathological examination.

1.3.4 Indices for Measurement.

Serum glutamate-pyruvate transaminase (ALT), glutamic-oxaloacetictransaminase (AST), histopathological examination of liver.

1.4 Measurement of ALT and AST.

1.4.1 Measuring Method.

Full-automatic biochemical analyzer or Reitman-Frankel method (reagentkit) can be selected.

1.4.1 Data Treatment and Result Assessment.

Variance analysis is used, but variance homogeneity test should beperformed first according to the procedures of variance analysis. If thevariance is homogeneous, F value is calculated. If F value is <F_(0.05),the conclusion is that the difference between means of different groupsis not significant. If F value is ≧F_(0.05) and P is ≦0.05, the methodof paired comparison of means between several experimental groups andone control group is used for statistical analysis. For data withabnormal distribution or variance inhomogeneity, appropriate conversionof variables is performed and the converted data are used forstatistical analysis after the requirement of normal or variancehomogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used for statistical analysis.

If ALT and AST of the test sample group are different significantly fromthose of the model control group, the results of ALT and AST can beassessed as positive respectively.

1.5 Histopathological Changes of Liver Diagnostic Criteria and ResultAssessment.

1.5.1 Experimental Materials.

The left lobe of rat liver is fixed with 10% formalin. The liver tissueis taken from the cross section of middle part of the left lobe of liverfor routine preparation of pathological section (paraffin embedding,H.E. strain).

1.5.2 Microscopic Examination.

Using the 40-fold objective to observe continuously the whole tissuesection, the pathological changes of cells are recorded beginning fromthe visual field of one end of the liver. The degenerative changes ofcentral liver cells of the lobe and necrosis of a few cells can be seen.The main types of pathological changes are ballooning degeneration,fatty degeneration, condensation of cytoplasm, hydropic degeneration andnecrosis of liver cells, etc.

1.5.3 Criteria for Rating.

Each pathological change accounting for the portion of area of visualfield in each visual field is recorded respectively and the total scoreof pathological changes in the visual fields observed is added up.

Ballooning degeneration of liver cells: (swelling of cells, a littlecytoplasm remains)

Roughly normal 0 points Liver cells with ballooning degeneration accountfor 1 point ¼ of whole visual field Liver cells with ballooningdegeneration account for 2 points ½ of whole visual field Liver cellswith ballooning degeneration account for 3 points ¾ of whole visualfield Liver cells with ballooning degeneration account for 4 pointswhole visual fieldFatty degeneration of liver cells: (distinctly demarcated fat dropvacuoles appear in cytoplasm of liver cells)

Roughly normal 0 points Liver cells with fatty degeneration account for1 point ¼ of whole visual field Liver cells with fatty degenerationaccount for 2 points ½ of whole visual field Liver cells with fattydegeneration account for 3 points ¾ of whole visual field Liver cellswith fatty degeneration account for 4 points whole visual fieldCondensation of cytoplasm: (eosinophilic stain is enhanced)

Roughly normal 0 points Liver cells with condensation of cytoplasmaccount for 1 point ¼ of whole visual field Liver cells withcondensation of cytoplasm account for 2 points 2/4 of whole visual fieldLiver cells with condensation of cytoplasm account for 3 points ¾ ofwhole visual field Liver cells with condensation of cytoplasm accountfor 4 points whole visual fieldHydropic degeneration:

No liver cell with hydropic change is seen 0 points Liver cells withhydropic degeneration account for 1 point ¼ of whole visual field Livercells with hydropic degeneration account for 2 points 2/4 of wholevisual field Liver cells with hydropic degeneration account for 3 points¾ of whole visual field Diffuse liver cells with hydropic degenerationaccount 4 points for whole visual fieldNecrosis of liver cells: (eosinophilic change of cytoplasm, coagulationnecrosis)

No necrotic cell is seen 0 points Sporadic necrotic cells account for ¼of whole visual field 1 point Necrotic cells account for 2/4 of wholevisual field 2 points Necrotic cells account for ¾ of whole visual field3 points Diffuse necrotic cells account for whole visual field 4 points

1.5.4 Data Treatment and Result Assessment.

Variance analysis is used, but variance homogeneity test should beperformed first according to the procedures of variance analysis. If thevariance is homogeneous, F value is calculated. If F value is <F_(0.05),the conclusion is that the difference between means of different groupsis not significant. If F value is ≧F_(0.05) and P is ≦0.05, the methodof paired comparison of means between several experimental groups andone control group is used for statistical analysis. For data withabnormal distribution or variance inhomogeneity, appropriate conversionof variables is performed and the converted data are used forstatistical analysis after the requirement of normal or variancehomogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used for statistical analysis.

Among the pathological changes of liver cells including ballooningdegeneration, fatty degeneration, cytoplasm condensation, hydropicdegeneration and necrosis of liver cells, if the necrosis of liver cellsin any dosage group of test sample is alleviated as compared with thatin the model control group with significant difference and other typesof pathological changes are alleviated significantly or have nosignificant difference as compared with those in the model controlgroup, the results of animal pathological experiment can be assessed aspositive.

If aggravation and alleviation of the 4 types of pathological changes ofliver cells, namely, ballooning degeneration, fatty degeneration,cytoplasm condensation and hydropic degeneration, are presentsimultaneously with significant difference and necrosis of liver cellsis alleviated in any one dosage group of the test sample withsignificant difference as compared with the model control group, thescores of various pathological changes and double of necrosis score areadded together. The total score is used for statistical analysis. If thetotal score has significant difference, the results of animalpathological experiment can be assessed as positive.

1.6 Assessment of Results.

It is expected that any one of the two blood biochemical indices, ALTand AST, and the result of pathological examination will be positive andthe test sample will be assessed as assisting in the protection againstchemical injury to the liver.

Protocol 2: Model of Alcoholic Injury of Liver

2.1 Principles.

After taking large amounts of ethyl alcohol, massive dehydroxylationcatalyzed by ethanol dehydrogenase causes disorder of tricarboxylic acidcycle and weakening of oxidation of fatty acid, thereby influencing fatmetabolism and precipitation of fat in liver cells. At the same time,ethyl alcohol can activate oxygen molecules and cause production ofoxygen free radicals, leading to lipid peroxidation of liver cellmembrane and depletion of reduced glutathione in body.

2.2 Experimental Animals.

Adult mice or rats of single sex. Each group consists of 8-12 rats(180-220 g) or 10-15 mice (18-22 g).

2.3 Experimental Methods and Procedures.

2.3.1 Dosage Groups and Duration of Administration of the Test Sample.

Three dosage groups, one blank control group and one model control groupare set. The dosage of one of the dosage groups is 10 times (mice) or 5times (rats) the recommended human dosage. A positive control group maybe set if necessary. Absolute ethyl alcohol (analytically pure) is usedto form model of liver injury. The concentration of absolute ethylalcohol is 50% (diluted with distilled water) and the dosage forintragastric administration to mice is 12-14 mL/kg BW (equivalent toethyl alcohol 6000-7000 mg/kg BW). The duration of administration of thetest sample is 30 days and can be prolonged to 45 days if necessary.

2.3.2 Route of Administration of the Test Sample.

The test sample is given intragastrically. If intragastricadministration is impossible, the test sample can be mixed in feed ordrinking water, and the feed intake and drinking water drunk of eachanimal is recorded.

2.3.3 Experimental Procedures.

The animals of the test sample groups are given intragastrically thetest sample every day and those of the blank control group and modelcontrol group are given distilled water. The animals are weighed twiceweekly and the dose of the test sample is adjusted according to bodyweight. At the time of completion of administration of the test sample,a single dose of 50% ethyl alcohol 12 mL/kg BW is given to the animalsof the model control group and three dosage groups, while the animals ofthe blank control group are given distilled water. After fasting for 16h, the animals are sacrificed for examination of various indices andhistopathological examination.

2.3.4 Indices for Examination.

Malondialdehyde (MDA), reduced glutathione (GSH), triglyceride (TG)content of liver.

2.4 Method for Measuring the Degradation Product of Lipid PeroxideMalondialdehyde (MDA) in Liver Homogenate.

2.4.1 Principle.

MDA is one of the final products of peroxidation of lipids of cellmembrane. Measuring MDA content can estimate indirectly the degree oflipid peroxidation. When MDA and thiobarbital are heated together inacidic condition, pink-colored complex is formed and its absorption peakis at 535 nm, from which the MDA content can be measured.

2.4.2 Instruments and Reagents.

Instruments: 721 spectrophotometer, sample micro-applicator, thermostatwater bath, ordinary centrifuge, mixing rotator, centrifuge tube withstopper, tissue homogenizer.

Reagents: 0.2M acetate buffer solution, pH 3.5:

0.2M acetic acid solution 185 mL 0.2 M sodium acetate solution  15 mL1 mmol/L tetraethoxyl propane (stock solution, kept at 4° C. for 3months), diluted with water to 40 nmol/mL just before use:

-   -   8.1% sodium dodecyl sulfate SDS    -   0.8% thiobarbital TBA    -   0.2 M phosphate buffer solution, pH 7.4        -   0.2 M disodium hydrogen phosphate 1920 mL        -   0.2 M potassium dihydrogen phosphate 480 mL

2.4.3 Experimental Procedures.

2.4.3.1 Preparation of Sample.

Tissue homogenate sample: certain quantity of the organ needed is rinsedwith normal saline, wiped to dry, weighed, minced and put intohomogenizer. 0.2 M phosphate buffer solution is added and the mixture ishomogenized at 2000 r/min for 10 s. The centrifugation is repeated 3times with 30 s intervals to form 5% tissue homogenate (W/V). Thehomogenate is centrifugalized at 3000 r/min for 5-10 min and thesupernatant is taken for measurement.

2.4.3.2 Measurement of the Sample.

Reagent Blank tube Sample tube Standard tube   5% tissue homogenate 0.1mL 40 nmol/mL tetraethoxyl 0.1 mL propane 8.1% SDS 0.2 mL 0.2 mL 0.2 mL0.2 M acetate buffer solution 1.5 mL 1.5 mL 1.5 mL 0.8% TBA 1.5 mL 1.5mL 1.5 mL H₂O 0.8 mL 0.7 mL 0.7 mL Mix to homogenize, boiling water bathfor 60 min protect from light, cooled with flowing water, colorimetry at532 nm2.4.3.3 Calculation.

${{Lipid}\mspace{14mu}{peroxide}\mspace{14mu}{content}\mspace{11mu}\left( {{{nmol}/{mg}}\mspace{14mu}{tissue}} \right)} = {{\frac{B - A}{F - A} \times C \times K} = {\frac{B - A}{F - A} \times 40 \times \frac{1}{0.05 \times 1000}}}$${{Lipid}\mspace{14mu}{peroxide}\mspace{14mu}{content}\mspace{11mu}\left( {{{nmol}/100}\mspace{11mu}{mg}\mspace{14mu}{protein}} \right)} = {{\frac{B - A}{F - A} \times C \times K} = {\frac{B - A}{F - A} \times 40 \times \frac{1}{0.05} \times \frac{1}{{Protein}\mspace{11mu}{({mg})/g}\mspace{14mu}{tissue}} \times 100}}$

A: absorbance of blank tube

B: absorbance of sample tube

F: absorbance of tetraethoxyl propane

C: concentration of tetraethoxyl propane (40 nmol/mL)

K: multiple of dilution

2.4.3.4 Data Treatment and Result Assessment.

The data are analyzed with variance analysis, but variance homogeneitytest should be performed first according to the procedures of varianceanalysis. If the variance is homogeneous, F value is calculated. If Fvalue is <F_(0.05), the conclusion is that the difference between meansof different groups is not significant. If F value is ≧F_(0.05) and P is≦0.05, the method of paired comparison of means between severalexperimental groups and one control group is used for statisticalanalysis. For data with abnormal distribution or variance inhomogeneity,appropriate conversion of variables is performed and the converted dataare used for statistical analysis after the requirement of normal orvariance homogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used for statistical analysis.

Assessment of Results

It is expected that the MDA content of test sample groups will besignificantly different from that of the model control group and, assuch, the result of this index will be assessed as positive.

2.5 Method for Measuring Reduced Glutathione (GSH) in Liver Homogenate.

2.5.1 Principle.

Reaction between GSH and 5,5′-dithionitroformic acid (DTNB) catalyzed byGSH-Px produces yellow-colored 5-thio-2-nitro-formic acid anion whichhas maximum absorption peak at wavelength of 423 nm. Measuring theconcentration of this ion can calculate GSH content.

2.5.2 Reagents.

-   -   0.9% normal saline    -   4% sulfosalicylic acid solution    -   0.1 mol/L PBS solution (pH=8.0)

Na₂HPO₄ 13.452 g KH₂PO₄  0.722 g

-   -   -   Distilled water ad 1000 mL.

    -   0.004% DTNB solution: DTNB 40 mg is dissolved in 1000 mL of 0.1        mol/L PBS solution (pH=8.0).

Sodium azide buffer solution.

NaN₃ 16.25 mg EDTA-Na₂ 7.44 mg Na₂HPO₄ 1.732 g NaH₂PO₄ 1.076 g

-   -   -   Distilled water ad 1000 mL. Small amount of HCl and NaOH are            used to regulate pH 7.0.        -   The solution is kept at 4° C.

Standard solution: Reduced GSH 15.4 mg is weighed and sodium azidebuffer solution is added to 50 mL to make the final concentration 1mmol/L. The solution is prepared just before use.

2.5.3 Methods.

2.5.3.1 Measurement of Sample.

Normal saline 5 mL is added to liver 0.5 g. The mixture is well groundto form fine thick liquid (10% liver homogenate). After homogenizing, 4%sulfosalicylic acid 0.5 mL is added to the homogenate 0.5 mL. Aftermixing, the mixture is centrifugalized at 3000 rpm for 10 min at roomtemperature and the supernatant is the sample.

Reagent Tube for measurement Blank tube Sample 0.5 mL — 4%sulfosalicylic acid — 0.5 mL DINB 4.5 mL 4.5 mLThe mixture is mixed, laid aside for 10 min at room temperature and itsabsorbance is measured at 412 nm.2.5.3.2 Standard Curve.

Reagent 1 2 3 4 5 6 1 mmol/L GSH 0 0.05 0.10 0.15 0.20 0.25 (mL) Normalsaline 0.50 0.45 0.40 0.35 0.20 0.25 (mL) DTNB (mL) 4.50 4.50 4.50 4.504.50 4.50 GSH content 0 100 200 300 400 500 (μmol/L)2.5.3.3 Calculation.

Sample GSH content (μmol/L liver tissue)=corresponding curveconcentration value (μmol/L)÷50 g/L

2.5.4 Data Treatment and Result Assessment.

The data are analyzed with variance analysis, but variance homogeneitytest is performed first according to the procedures of varianceanalysis. If the variance is homogeneous, F value is calculated. If Fvalue is <F_(0.05), the conclusion is that the difference between meansof different groups is not significant. If F value is ≧F_(0.05) and P is≦0.05, the method of paired comparison of means between severalexperimental groups and one control group is used for statisticalanalysis. For data with abnormal distribution or variance inhomogeneity,appropriate conversion of variables is performed and the converted dataare used for statistical analysis after the requirement of normal orvariance homogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used.

Assessment of Results

It is expected that the reduced GSH content of the test sample groupwill be significantly different from that of the model control groupand, as such, the result of this index is assessed as positive.

2.6 Method for Measuring Triglyceride (TG) in Liver Homogenate.

2.6.1 Measuring Method.

Triglyceride measurement reagent kit (glycerophosphoric acid oxidaseperoxidase method) is used to measure the triglyceride content in 10%liver homogenate. Same as the method of measuring serum triglyceride,equal amount of 10% liver homogenate is used instead of serum and themeasurement is performed according to the description of operation. Theresult of measurement is expressed as mmol/g liver weight.

2.6.2 Data Treatment and Result Assessment.

The data are treated with variance analysis, but variance homogeneitytest is performed first according to the procedures of varianceanalysis. If the variance is homogeneous, F value is calculated. If Fvalue is <F0.05, the conclusion is that the difference between means ofdifferent groups is not significant. If F value is ≧F_(0.05) and P is≦0.05, the method of paired comparison of means between severalexperimental groups and one control group is used for statisticalanalysis. For data with abnormal distribution or variance inhomogeneity,appropriate conversion of variables is performed and the converted dataare used for statistical analysis after the requirement of normal orvariance homogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used for statistical analysis.

Assessment of Results

It is expected that the TG of the test sample group will besignificantly different from that of the model control group and, assuch, the result of this index will be assessed as positive.

2.7 Histopathological Changes of Liver, Diagnostic Criteria and ResultAssessment.

2.7.1 Experimental Materials.

Cross section at middle part of left lobe of liver is performed fortaking examination material. Frozen section is made and stained withSudan III staining.

2.7.2 Microscopic Examination.

The pathological changes of the cells are recorded beginning from thevisual field at one end of the liver. 40-fold objective is used forcontinuous observation of whole tissue section. The main objects forobservation are distribution, range and area of fat drops in liver.

2.7.3 Criteria for Rating.

The fat drops in liver cells are sporadic and scarce 0 points The livercells containing fat drops do not exceed ¼ 1 point  The liver cellscontaining fat drops do not exceed ½ 2 points The liver cells containingfat drops do not exceed ¾ 3 points The liver tissue is almost replacedby fat drops 4 points

2.7.4 Data Treatment and Result Assessment.

Variance analysis is used, but variance homogeneity test should beperformed first according to the procedures of variance analysis. If thevariance is homogeneous, F value is calculated. If F value is <F_(0.05),the conclusion is that the difference between means of different groupsis not significant. If F value is ≧F_(0.05) and P is ≦0.05, the methodof paired comparison of means between several experimental groups andone control group is used for statistical analysis. For data withabnormal distribution or variance in homogeneity, appropriate conversionof variables is performed and the converted data are used forstatistical analysis after the requirement of normal or variancehomogeneity is fulfilled. If the purpose of normal or variancehomogeneity is still not achieved after conversion of variables, ranktest is used for statistical analysis.

It is expected that the fatty degeneration in any dosage group of testsample will be alleviated as compared with the model control group withstatistical difference and, as such, the result will be assessed aspositive.

2.8 Assessment of the Results.

It is expected that the following conditions will be fulfilled and, assuch, the test sample would be assessed as assisting in protectionagainst alcoholic liver injury:

(a) The results of examination of 3 indices, namely, liver MDA, reducedGSH and TG, are positive.

(b) Any two of the 3 indices, namely, liver MDA, reduced GSH and TG, arepositive and the results of histopathological examination are positive.

1. A composition for improving liver health comprising about 150 mg-500mg of wasabi root fiber powder, about 200 mg-1000 mg of artichoke leafextract, and about 100 mg-500 mg of notoginseng extract, wherein thecomposition has phase II enzyme induction activity.
 2. The compositionof claim 1 wherein the composition further comprises about 150mg-1000mgschisandra berry extract, and about 20 mg-500 mg spinach dehydrate. 3.The composition of claim 1 wherein the composition further comprisesabout 150 mg-1000mg kudzu root extract, and about 20mg-500mg oregano. 4.The composition of claim 1, further comprising about 150 mg-1000 mgkudzu root and about 20 mg-500 mg spinach dehydrate.